Methods for detecting lacosamide by mass spectrometry

ABSTRACT

Provided are methods for determining the amount of lacosamide in a sample using mass spectrometry. The methods generally involve ionizing lacosamide in a sample and detecting and quantifying the amount of the ion to determine the amount of lacosamide in the sample.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/927,769, filed Jul. 13, 2020, now U.S. Pat. No. 11,360,061, which isa continuation of U.S. application Ser. No. 16/715,823, filed Dec. 16,2019, now U.S. Pat. No. 10,712,319, which is a continuation of U.S.application Ser. No. 15/787,439, filed Oct. 18, 2017, now U.S. Pat No.10,509,016, which is a continuation of U.S. application Ser. No.15/388,741, filed Dec. 22, 2016, now U.S. Pat. No. 9,823,227, which is acontinuation of U.S. application Ser. No. 14/319,829, filed Jun. 30,2014, now U.S. Pat. No. 9,530,635, which is a continuation of U.S.application Ser. No. 13/339,267, filed Dec. 28, 2011, now U.S. Pat. No.8,779,355, each of which is incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

The invention relates to the detection of lacosamide. In a particularaspect, the invention relates to methods for detecting lacosamide bymass spectrometry.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

Lacosamide (also known as Vimpat™, erlosamide, and harkoseride) is afunctionalized amino acid that is used as an adjunctive therapy thetreatment of partial-onset seizures (Halasz et al., Epilepsia,50(3):443-453, 2009). Lacosamide is also being investigated as atreatment for diabetic neuropathic pain (Ziegler et al. Diabetes Care.2010 April; 33(4):839-41). Lacosamide has dual mechanisms of action. Inone mechanism, lacosamide selectively enhances slow inactivation ofvoltage-gated sodium channels, which in turn stabilizes hyperexcitableneuronal membranes and inhibits neuronal firing (Sheets et al., JPharmacol Exp Ther. 2008 July; 326(1):89-99; Errington et al., MolPharmacol. 2008 January; 73(1):157-69). In another mechanism of action,lacosamide modulates collapsin response mediator protein-2 (CRMP-2), aprotein that has altered expression in epilepsy and otherneurodegenerative diseases (Cada et al., Hospital Pharmacy 2009 June;44(6), 497-508).

Assays for lacosamide blood levels have been developed and are used bypatients and physicians to optimize therapeutic dosages.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the amount oflacosamide in a sample by mass spectrometry, including tandem massspectrometry.

In one aspect, methods are provided for determining the amount oflacosamide in a sample. Methods of this aspect include: (a) ionizinglacosamide from the sample to produce one or more lacosamide ionsdetectable by mass spectrometry, wherein the ions comprise one or moreions selected from the group consisting of ions with a mass/charge ratioof 251.0±0.5, 91.0±0.5, and 108.0±0.5; (b) determining the amount of oneor more lacosamide ions by mass spectrometry; and (c) using the amountof the lacosamide ion(s) to determine the amount of lacosamide in thesample. In some embodiments, the mass spectrometry is tandem massspectrometry. In some embodiments, the methods further include purifyinglacosamide in the sample prior to mass spectrometry. In someembodiments, purifying includes purifying with liquid chromatography. Inrelated embodiments, the liquid chromatography comprises highperformance liquid chromatography (HPLC). In some embodiments, purifyingincludes one or more purification steps followed by liquidchromatography. In related embodiments, the one or more purificationsteps prior to liquid chromatography include protein precipitation. Insome embodiments, the sample is generated by subjecting a body fluid toone or more processing steps. In some embodiments, the sample isgenerated by subjecting plasma or serum to one or more processing steps.In some embodiments, ionizing includes generating a lacosamide precursorion with a mass/charge ratio of 251.0±0.5, and generating one or morelacosamide fragment ions selected from the group consisting of ions witha mass/charge ratio of 91.0±0.5 and 108.0±0.5. In some embodiments,ionizing is conducted in positive ion mode. In some embodiments, themethod has a lower limit of quantitation within the range of 20 μg/mland 0.5 μg/ml, inclusive.

In a second aspect, methods are provided for determining the amount oflacosamide in a body fluid sample by tandem mass spectrometry. Methodsof this aspect include: (a) ionizing lacosamide, purified from the bodyfluid sample by liquid chromatography, to generate a lacosamideprecursor ion having a mass/charge ratio of 251.0±0.5; (b) producing oneor more lacosamide fragment ions of the lacosamide precursor ion,wherein at least one of the one or more lacosamide fragment ionscomprise an ion selected from the group of ions having a mass/chargeratio of 91.0±0.5 and 108.0±0.5; and (c) determining the amount of oneor more of the ions generated in step (b) or (c) or both and relatingthe determined ions to the amount of lacosamide in the body fluidsample. In some embodiments, the methods have a lower limit ofquantitation within the range of 20 μg/ml and 0.5 μg/ml, inclusive. Insome embodiments, the liquid chromatography includes high performanceliquid chromatography (HPLC). In some embodiments, the lacosamidepurified from a body fluid sample is purified by one or morepurification steps followed by liquid chromatography. In relatedembodiments, the one or more purification steps prior to liquidchromatography include protein precipitation. In some embodiments, thebody fluid sample includes plasma or serum. In some embodiments,ionization is conducted in positive ion mode.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “processing step” refers to any sample handlingstep without limitation, including, for example: heating, cooling,centrifugation, or purification by any method known in the art. Inparticular, purification steps may include one or more of filtration,protein precipitation, liquid-liquid extraction, solid-phase extraction,liquid chromatography (including high performance liquidchromatography), and the like.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedlacosamide parent or daughter ions by mass spectrometry. Relativereduction as this term is used does not require that any substance,present with the analyte of interest in the material to be purified, isentirely removed by purification.

As used herein, the term “sample” refers to any sample that may containlacosamide. As used herein, the term “body fluid” means any fluid thatcan be isolated from the body of an individual. For example, “bodyfluid” may include blood, plasma, serum, bile, saliva, urine, tears,perspiration, and the like.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and high turbulence liquidchromatography (HTLC).

As used herein, the term “high performance liquid chromatography” or“HPLC” refers to liquid chromatography in which the degree of separationis increased by forcing the mobile phase under pressure through astationary phase on a support matrix, typically a densely packed column.

As used herein, the term “high turbulence liquid chromatography” or“HTLC” refers to a form of chromatography that utilizes turbulent flowof the material being assayed through the column packing as the basisfor performing the separation. HTLC has been applied in the preparationof samples containing two unnamed drugs prior to analysis by massspectrometry. See, e.g., Zimmer et al., J. Chromatogr. A 854: 23-35(1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368, 5,795,469, and5,772,874, which further explain HTLC. Persons of ordinary skill in theart understand “turbulent flow”. When fluid flows slowly and smoothly,the flow is called “laminar flow”. For example, fluid moving through anHPLC column at low flow rates is laminar. In laminar flow the motion ofthe particles of fluid is orderly with particles moving generally instraight lines. At faster velocities, the inertia of the water overcomesfluid frictional forces and turbulent flow results. Fluid not in contactwith the irregular boundary “outruns” that which is slowed by frictionor deflected by an uneven surface. When a fluid is flowing turbulently,it flows in eddies and whirls (or vortices), with more “drag” than whenthe flow is laminar. Many references are available for assisting indetermining when fluid flow is laminar or turbulent (e.g., TurbulentFlow Analysis: Measurement and Prediction, P. S. Bernard & J. M.Wallace, John Wiley & Sons, Inc., (2000); An Introduction to TurbulentFlow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 35 μm, such as greater than about 50 μm.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means±10%. In apreferred embodiment the analytical column contains particles of about 5μm in diameter.

As used herein, the term “on-line” or “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500,entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat.No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;”U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced PhotolabileAttachment And Release For Desorption And Detection Of Analytes;” Wrightet al., Prostate Cancer and Prostatic Diseases 2:264-76 (1999); andMerchant and Weinberger, Electrophoresis 21:1164-67 (2000).

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber. As the droplets get smaller the electrical surface chargedensity increases until such time that the natural repulsion betweenlike charges causes ions as well as neutral molecules to be released.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. Robb, D. B., Covey, T. R. and Bruins,A. P. (2000): See, e.g., Robb et al., Atmospheric pressurephotoionization: An ionization method for liquid chromatography-massspectrometry. Anal. Chem. 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, the term “lower limit of quantification”, “limit ofquantitation” or “LOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible and is calculated as the meanplus 10 standard deviations (SD).

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as four standarddeviations from the zero concentration.

As used herein, an “amount” of lacosamide in a body fluid sample refersgenerally to an absolute value reflecting the mass of lacosamidedetectable in volume of body fluid. However, an amount also contemplatesa relative amount in comparison to another lacosamide amount. Forexample, an amount of lacosamide in a body fluid can be an amount whichis greater than a control or normal level of lacosamide normallypresent.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show exemplary chromatograms of 91 m/z fragment ionsfrom lacosamide and lacosamide-D3, respectively. FIG. 1C is an overlayof exemplary chromatograms of 91.0 m/z and 108.0 m/z fragment ions fromlacosamide and 91 m/z fragment ions from lacosamide-D3. FIG. 1D is achart showing the chromatogram intensities of 91.0 m/z and 108.0 m/zfragment ions from lacosamide and 91 m/z fragment ions fromlacosamide-D3. Details are discussed in Example 3.

FIG. 2 shows Q1 full scan spectra for lacosamide. Details are discussedin Example 3

FIG. 3A shows Q3 product ion scan spectra for fragmentation oflacosamide with a mass/charge ratio of 251.0±0.5 at a collision energyof 20 V. FIG. 3B shows Q3 product ion scan spectra for the samelacosamide precursor ion at a collision energy of 35 V. Details arediscussed in Example 3.

FIG. 4 shows the linearity of the quantitation of lacosamide in seriallydiluted stock samples using an LC-MS/MS assay. Details are described inExample 6.

FIG. 5 shows a graph comparing lacosamide values in spiked samples ofserum, EDTA, and heparin specimen types. Details are discussed inExample 7.

DETAILED DESCRIPTION OF THE INVENTION

Methods of the present invention are described for measuring the amountof lacosamide in a sample. More specifically, mass spectrometric methodsare described for detecting and quantifying lacosamide in a sample. Themethods may utilize liquid chromatography (LC), preferably HPLC, toperform a purification of selected analytes, and combine thispurification with unique methods of mass spectrometry (MS), therebyproviding a high-throughput assay system for detecting and quantifyinglacosamide in a sample. The embodiments are particularly well suited forapplication in large clinical laboratories for automated lacosamideassay.

Suitable samples for use in methods of the present invention include anysample that may contain the analyte of interest. In some preferredembodiments, a sample is a biological sample; that is, a sample obtainedfrom any biological source, such as an animal, a cell culture, an organculture, etc. In certain preferred embodiments, samples are obtainedfrom a mammalian animal, such as a dog, cat, horse, etc. Particularlypreferred mammalian animals are primates, most preferably male or femalehumans. Particularly preferred samples include bodily fluids such asblood, plasma, serum, saliva, cerebrospinal fluid, or a tissue sample.Such samples may be obtained, for example, from a patient; that is, aliving person, male or female, presenting oneself in a clinical settingfor diagnosis, prognosis, or treatment of a disease or condition. Thetest sample is preferably obtained from a patient, for example, bloodserum or plasma.

The present invention contemplates kits for a lacosamide quantitationassay. A kit for a lacosamide quantitation assay of the presentinvention may include a kit comprising an internal standard in amountssufficient for at least one assay. Typically, the kits will also includeinstructions recorded in a tangible form (e.g., contained on paper or anelectronic medium) for using the packaged reagents for use in ameasurement assay for determining the amount of lacosamide.

Calibration and QC pools for use in embodiments of the present inventioncan be prepared using plasma or serum that has been spiked withlacosamide. All sources of human or non-human plasma or serum to be usedin calibration and QC pools should be checked to ensure that they do notcontain measurable amounts of lacosamide.

Sample Preparation for Mass Spectrometry

Biological and non-biological samples at or near room temperaturetypically do not require any temperature adjustment. Frozen orrefrigerated samples (including controls) are typically thawed andbrought to or near room temperature rapidly. Internal standard may beadded to frozen samples once they are thawed.

Optionally, samples may be prepared for mass spectrometry by subjectingthe sample to one or more methods to enrich lacosamide relative to othercomponents in the sample (e.g. protein). Various methods may be used toenrich lacosamide relative to other components in the sample (e.g.protein) prior mass spectrometry, including for example, liquid-liquidextraction (e.g., ethyl acetate extraction and methanol extraction),solid-phase extraction, liquid chromatography, filtration,centrifugation, thin layer chromatography (TLC), electrophoresisincluding capillary electrophoresis, affinity separations includingimmunoaffinity separations, and the use of chaotropic agents or anycombination of the above or the like.

Protein precipitation is one preferred method of preparing a testsample, especially a biological test sample, such as serum or plasma.Such protein purification methods are well known in the art, forexample, Polson et al., Journal of Chromatography B 785:263-275 (2003),describes protein precipitation techniques suitable for use in methodsof the present invention. Protein precipitation may be used to removemost of the protein from the sample leaving lacosamide in thesupernatant. The samples may be centrifuged to separate the liquidsupernatant from the precipitated proteins; alternatively the samplesmay be filtered, for example through a glass fiber filter, to removeprecipitated proteins. The resultant supernatant or filtrate may then beapplied directly to mass spectrometry analysis; or alternatively toliquid chromatography and subsequent mass spectrometry analysis. Incertain embodiments, the use of protein precipitation such as forexample, methanol protein precipitation, may obviate the need for highturbulence liquid chromatography (HTLC) or other on-line extractionprior to mass spectrometry or HPLC and mass spectrometry.

Accordingly, in some embodiments, the method involves (1) performing aprotein precipitation of the sample of interest; and (2) loading thesupernatant directly onto the LC-mass spectrometer without using on-lineextraction or high turbulence liquid chromatography (HTLC).

In some embodiments, HTLC, alone or in combination with one or morepurification methods, may be used to purify lacosamide prior to massspectrometry. In such embodiments samples may be extracted using an HTLCextraction cartridge which captures the analyte, then eluted andchromatographed on a second HTLC column or onto an analytical HPLCcolumn prior to ionization. Because the steps involved in thesechromatography procedures may be linked in an automated fashion, therequirement for operator involvement during the purification of theanalyte can be minimized. This feature may result in savings of time andcosts, and eliminate the opportunity for operator error.

One means of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Liquid chromatography,including high-performance liquid chromatography (HPLC), relies onrelatively slow, laminar flow technology. Traditional HPLC analysisrelies on column packing in which laminar flow of the sample through thecolumn is the basis for separation of the analyte of interest from thesample. The skilled artisan will understand that separation in suchcolumns is a diffusion process and may select HPLC instruments andcolumns that are suitable for use with lacosamide. The chromatographiccolumn typically includes a medium (i.e., a packing material) tofacilitate separation of chemical moieties (i.e., fractionation). Themedium may include minute particles. The particles include a bondedsurface that interacts with the various chemical moieties to facilitateseparation of the chemical moieties. One suitable bonded surface is ahydrophobic bonded surface such as an alkyl bonded surface. Alkyl bondedsurfaces may include C-4, C-8, C-12, or C-18 bonded alkyl groups.Alternatively, a suitable surface is a base-deactivated biphenyl silicasurface, such as found in a Pinnacle® DB Biphenyl HPLC column. Thechromatographic column includes an inlet port for receiving a sampledirectly or indirectly from coupled SPE column and an outlet port fordischarging an effluent that includes the fractionated sample.

In one embodiment, the sample may be applied to the column at the inletport, eluted with a solvent or solvent mixture, and discharged at theoutlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytyptic(i.e. mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one embodiment, HPLC is conducted with a hydrophobic columnchromatographic system. In certain preferred embodiments, a Pinnacle® DBBiphenyl analytical column (50×2.1 mm, 5 μl column or equivalent) isused. In certain preferred embodiments, HTLC and/or HPLC are performedusing HPLC Grade 0.1% formic acid in methanol and Grade 0.1% formic acidin water as the mobile phases.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

Detection and Quantitation by Mass Spectrometry

In various embodiments, lacosamide present in a test sample may beionized by any method known to the skilled artisan. Mass spectrometry isperformed using a mass spectrometer, which includes an ion source forionizing the fractionated sample and creating charged molecules forfurther analysis. For example ionization of the sample may be performedby electron ionization, chemical ionization, electrospray ionization(ESI), photon ionization, atmospheric pressure chemical ionization(APCI), photoionization, atmospheric pressure photoionization (APPI),fast atom bombardment (FAB), liquid secondary ionization (LSI), matrixassisted laser desorption ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, surface enhanced laserdesorption ionization (SELDI), inductively coupled plasma (ICP) andparticle beam ionization. The skilled artisan will understand that thechoice of ionization method may be determined based on the analyte to bemeasured, type of sample, the type of detector, the choice of positiveversus negative mode, etc.

In preferred embodiments, lacosamide is ionized by ESI in positive ionmode. In related preferred embodiments, lacosamide ion is in a gaseousstate and the inert collision gas is argon or nitrogen; preferablynitrogen.

In mass spectrometry techniques generally, after the sample has beenionized the positively charged or negatively charged ions therebycreated may be analyzed to determine a mass-to-charge ratio. Suitableanalyzers for determining mass-to-charge ratios include quadrupoleanalyzers, ion traps analyzers, magnetic and electric sector analyzers,and time-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected, i.e. usinga selective ion monitoring mode (SIM), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM). Preferably, the mass-to-chargeratio is determined using a quadrupole analyzer. For example, in a“quadrupole” or “quadrupole ion trap” instrument, ions in an oscillatingradio frequency field experience a force proportional to the DCpotential applied between electrodes, the amplitude of the RF signal,and the mass/charge ratio. The voltage and amplitude may be selected sothat only ions having a particular mass/charge ratio travel the lengthof the quadrupole, while all other ions are deflected. Thus, quadrupoleinstruments may act as both a “mass filter” and as a “mass detector” forthe ions injected into the instrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion is subsequentlyfragmented to yield one or more fragment ions (also called daughter orproduct ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular mass/chargeover a given range (e.g., 100 to 1000 amu). The results of an analyteassay, that is, a mass spectrum, may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, standards may be run with the samples,and a standard curve constructed based on ions generated from thosestandards. Using such a standard curve, the relative abundance of agiven ion may be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of lacosamide.Methods of generating and using such standard curves are well known inthe art and one of ordinary skill is capable of selecting an appropriateinternal standard. For example, an isotopically labeled lacosamide maybe used as an internal standard; in certain preferred embodiments thestandard is lacosamide-D3. Numerous other methods for relating theamount of an ion to the amount of the original molecule will be wellknown to those of ordinary skill in the art.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activation dissociation is oftenused to generate the fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In particularly preferred embodiments, lacosamide is detected and/orquantified using MS/MS as follows. The samples are subjected to proteinprecipitation followed by liquid chromatography, preferably HPLC; theflow of liquid solvent from the chromatographic column enters the heatednebulizer interface of an MS/MS analyzer; and the solvent/analytemixture is converted to vapor in the heated tubing of the interface. Theanalyte (e.g., lacosamide), contained in the nebulized solvent, isionized by the corona discharge needle of the interface, which applies alarge voltage to the nebulized solvent/analyte mixture. The ions, e.g.precursor ions, pass through the orifice of the instrument and enter thefirst quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters,allowing selection of ions (i.e., selection of “precursor” and“fragment” ions in Q1 and Q3, respectively) based on their mass tocharge ratio (m/z). Quadrupole 2 (Q2) is the collision cell, where ionsare fragmented. The first quadrupole of the mass spectrometer (Q1)selects for ions with the mass to charge ratios of a particularlacosamide precursor ion. Precursor ions with the correct mass/chargeratios are allowed to pass into the collision chamber (Q2), whileunwanted ions with any other mass/charge ratio collide with the sides ofthe quadrupole and are eliminated. Precursor ions entering Q2 collidewith a neutral collision gas, e.g. nitrogen, and fragment. This processis called collision activated dissociation (CAD). The lacosamidefragment ions are passed into quadrupole 3 (Q3), where particularlacosamide fragment ions are allowed to pass to the detector while otherions are eliminated.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably positive ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of lacosamide that maybe used for selection in quadrupole 3 (Q3).

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, are measured and thearea or amplitude is correlated to the amount of the analyte ofinterest. In certain embodiments, the area under the curves, oramplitude of the peaks, for fragment ion(s) and/or precursor ions aremeasured to determine the amount of lacosamide. As described above, therelative abundance of a given ion may be converted into an absoluteamount of the original analyte, e.g., lacosamide, using calibrationstandard curves based on peaks of one or more ions of an internalmolecular standard, such as lacosamide-D3.

The following examples serve to illustrate the invention. These examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1: Sample (Plasma and Serum) and Reagent Preparation

Plasma samples were prepared by collecting blood in a Vacutainer® tubewith sodium heparin and refrigerating to about 2° C. to 8° C. Sampleswere then centrifuged (about 2200-2500 rpm, about 800-1000 g) for about8 to 10 minutes while maintaining refrigeration at about 2° C. to 8° C.The supernatants from the centrifugation were poured off and collectedfor later analysis.

Serum samples were prepared by collecting blood in a Vacutainer® tubewith no additives and allowed to clot for 20 to 30 minutes whilerefrigerated at about 2° C. to 8° C. The samples were then centrifuged(about 2200-2500 rpm, about 800-1000 g) for about 8 to 10 minutes whilemaintaining refrigeration at about 2° C. to 8° C. The resulting serumwas then transferred as above for plasma.

Lacosamide and Lacosamide-D3 (internal standard) stock solutions wereprepared. Lacosamide or lacosamide-D3 powders were mixed with methanolin separate glass vials. Aliquots of stock solutions were kept frozen.

Internal standard working solution of lacosamide-D3 (5 μg/ml) wasprepared by diluting lacosamide-D3 stock solution in methanol to thedesired concentration. Standard working solution of lacosamide (200μg/ml) was prepared by diluting lacosamide stock solution in 50%methanol to the desired concentration. Serum samples spiked with variousconcentrations of lacosamide were prepared by diluting the 200 μg/mllacosamide standard working solution with drug-free serum. For example,serum having 20 μg/ml of lacosamide was prepared by diluting thelacosamide working standard 1:10 in drug-free serum. The followingworking stocks of lacosamide in serum were prepared: 20 μg/ml (1:10), 10μg/ml (1:20), 8 μg/ml (1:25), 4 μg/ml (1:50), 2 μg/ml (1:100), 1 μg/ml(1:200), and 0.5 μg/ml (1:400).

Low level (about 2.5 μg/ml), medium level (about 5 μg/ml), and highlevel (about 12 μg/ml) lacosamide controls custom ordered from UTAKLaboratories were also used. The three levels of lacosamide controlswere mixed from drug-free serum (Bio-Rad #456) spiked with stocksolutions of lacosamide and lacosamide-D3.

Example 2: Extraction of Lacosamide from Plasma and Serum using LiquidChromatography

Liquid chromatography (LC) samples were prepared by thawing standards,controls, and patient samples to room temperature. Proteins wereprecipitated from 100 μl of each of the standards, controls, and patientsamples by adding 200 μl methanol and 20 μl of internal standard,vortexing for about 30 seconds, and then centrifuging for about 5minutes at about 14,000 g. The clear supernatant was then removed andpoured into the autosampler vials with flat bottom glass inserts forinsertion into an autosampler cooling unit.

Sample injection was performed with a Agilent 1200 series pump systemoperating in laminar flow mode using Analyst v. 1.5 or newer software.50% methanol in water was used as an autosampler wash solution.

The HPLC system automatically injected about 10 μL of the above preparedsamples standards and controls into a Restek guard column (catalog#940950212) followed by an HPLC analytical column (Pinnacle® DBBiphenyl, 50×2.1 mm, 5 μm column).

A binary HPLC gradient was applied to the analytical column to separatelacosamide from other analytes contained in the sample. Mobile phase Awas 0.1% formic acid in methanol and mobile phase B was 0.1% formic acidin water. A gradient program was used to elute and separate thecomponents from each other and reduce the presence of compounds likelyto cause ion suppression. First, a mixture of 10% mobile phase A/90%mobile phase B was applied to the column for 0.1 minutes. Then, thesolvent mixture was stepped to 70% mobile phase A/30% mobile phase B for0.1 minutes. This was followed by a linear ramp to 90% mobile phaseA/10% mobile phase B over 1.7 minutes, during which time the analyteeluted from the column. Finally, the solvent mixture was stepped back to10% mobile phase A/90% mobile phase B for 0.1 minutes to flush thecolumn. The gradient times, flow rates and solution percentages arelisted in Table 1 below.

TABLE 1 Gradient Program Total Time Flow Rate A (%) B (%) Step (min)(μl/min) [Organic] [Aqueous] Transition 0 0.10 750 10 90 Step 1 0.20 75070 30 Step 2 1.90 750 90 10 Ramp 3 2.00 750 10 90 StepThe separated sample was then subjected to MS/MS for quantitation oflacosamide.

Example 3: Detection and Quantitation of Lacosamide by MS/MS

MS/MS was performed using an API 3200 system (Applied Biosystems). Thesoftware program Analyst 1.5 from Applied Biosystems was used in theExamples described herein. Liquid solvent/analyte exiting the analyticalHPLC column flowed to the electro-spray ionization interface of the API3200 system where the analyte was ionized in positive ion mode.

Ions passed to the first quadrupole (Q1), which selected ions with amass to charge ratio of 251.0±0.5 m/z. Ions entering Quadrupole 2 (Q2)collided with nitrogen gas to generate ion fragments, which were passedto quadrupole 3 (Q3) for further selection. Simultaneously, the sameprocess using isotope dilution mass spectrometry was carried out with aninternal standard, lacosamide-D3. A second transition is monitored toprovide a qualifying ion ratio to ensure analyte identity. The masstransitions listed in Table 2 were used for detection and quantitationduring validation on positive polarity.

TABLE 2 Mass Transitions for Lacosamide Q3 qualifier Substance Q1 (m/z)Q3 (m/z) and CE (V) (m/z) and CE (V) Lacosamide 251.0 91.0 (at 35 V)108.0 (at 20 V) Lacosamide-D3 254.0 91.0 (at 35 V) —

Exemplary chromatograms for lacosamide and lacosamide-D3 (internalstandard) are found in FIGS. 1A, 1B, and 1C.

Exemplary precursor ion spectra for lacosamide are found in FIG. 2 .Exemplary fragmentation spectra of the lacosamide precursor ion with m/zof 251.0±0.5 are found in FIGS. 3A (collision energy of 20 V) and 3B(collision energy of 35 V).

Example 4: Intra-assay and Inter-assay Precision and Accuracy

Three quality control (QC) pools were prepared in the laboratory fromdrug-free serum, spiked with lacosamide to a concentration of 2.5, 5.0,and 15.0 μg/mL.

Five aliquots from each of the three QC pools were analyzed in a singleassay, and each single assay repeated five times to determine theoverall coefficient of variability (overall CV (%)) of a sample withinan assay. All three controls (low, medium and high) met the acceptablereproducibility requirements of <10% CV. The sigma values on allcontrols were greater than 3.0, giving an acceptable precision. TheProcess Sigma based on the SD and % CV and the defined allowable totalerror (TEa) was calculated as:Sigma (for precision only)=TEa (units)/SD (units)=TEa %/CV %

Calculated total error (TEc) was defined as a combination of imprecisionand inaccuracy (or bias) and was calculated asTEc=|bias|+m*SD,where the multiplier “m” may have values of 2, 3, 4, even up to 6 (forSix Sigma performance) and SD is the total standard deviation of themethod at or near the concentration where the bias was determined.Performance requirements and acceptability requirements may be derivedfrom TEa. Precision, including Intra-Assay and Inter-Assay, the Total SD(or Total CV) must be <TEa/3.

The resulting 25 data points and summary of their analysis are presentedin Table 2.

TABLE 2 Intra-Assay Variation and Accuracy for Laboratory Control Run 1Run 2 Run 3 Run 4 Run 5 Level 1 Sample 1 2.57 2.60 2.46 2.47 2.52 Sample2 2.66 2.50 2.61 2.53 2.46 Sample 3 2.55 2.58 2.57 2.47 2.49 Sample 42.61 2.56 2.61 2.54 2.44 Sample 5 2.62 2.64 2.59 2.55 2.59 Count 5 5 5 55 Average 2.60 2.58 2.57 2.51 2.50 In-Run SD 0.04 0.05 0.06 0.04 0.06Level 2 Sample 1 5.21 5.14 4.89 5.07 5.14 Sample 2 5.14 5.17 4.88 4.624.97 Sample 3 5.08 5.32 4.88 4.74 5.16 Sample 4 5.18 5.07 5.01 4.97 4.91Sample 5 5.23 5.15 4.90 4.89 4.97 Count 5 5 5 5 5 Average 5.17 5.17 4.914.86 5.03 In-Run SD 0.06 0.09 0.06 0.18 0.11 Level 3 Sample 1 16.3015.40 16.40 15.30 15.10 Sample 2 16.20 15.90 16.20 15.80 15.80 Sample 316.20 16.30 15.80 15.50 14.30 Sample 4 16.10 16.00 15.70 16.00 14.80Sample 5 16.20 15.90 16.20 15.90 14.70 Count 5 5 5 5 5 Average 16.2015.90 16.06 15.70 14.94 In-Run SD 0.07 0.32 0.30 0.29 0.56 Summary Level1 Level 2 Level 3 Count 25 25 25 Grand Mean 2.55 5.03 15.76 Pooled WR SD0.05 0.11 0.35 Pooled WR CV 2.03% 2.18% 2.19% Overall SD 0.06 0.16 0.55Overall CV 2.43% 3.27% 3.49% Sigma Overall 10.31 7.63 7.15

Thirty-five aliquots from each of three additional QC poolscustom-ordered from UTAK Laboratories at concentrations of 2.5, 5.0, and12.0 μg/ml were assayed over three separate days (ten aliquots in twoassays on day 1, fifteen aliquots in three assays on day 2, and tenaliquots in two assays on day 3) to determine the coefficient ofvariability (CV (%)) between assays. All controls met the acceptablereproducibility requirements of <10% CV. The results of these assays arepresented in Table 3.

TABLE 3 Inter-Assay Variation and Accuracy for UTAK Controls Level ILevel II Level III (2.5 μg/mL) (5.0 μg/mL) (12.0 μg/mL) Mean 2.89 5.7412.21 Standard Deviation 0.20 0.45 0.85 CV (%) 6.89% 7.85% 6.94%

Example 5: Analytical Sensitivity: Limit of Detection (LOD) and LowerLimit of Quantitation (LOQ)

The lower limit of quantitation (LOQ) is a measurement of selectivity,the ability of an analytical method to differentiate and quantify theanalyte in the presence of other components in the sample. The LOQ wasdetermined by assaying 20 replicates of serum diluent (i.e., blank). TheLOQ was calculated as the mean of the blank assays plus 10 standarddeviations (SD). The statistical LOQ for the lacosamide assay wasdetermined to be 1 ng/mL.

The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as four standarddeviations (SD) from the mean value of measuring zero concentration. Todetermine the LOD for the lacosamide assay, 20 replicates of serumdiluent (i.e., blank) were statistically analyzed. The LOD for thelacosamide assay was determined to be about 0.5 ng/mL.

Example 6: Assay Reportable Range and Linearity

To establish the linearity of lacosamide detection in the assay, sevenstandards with concentrations of 0.50 μg/ml, 1.00 μg/ml, 2.00 μg/ml,4.00 μg/ml, 8.00 μg/ml, 10.00 μg/ml, and 20 μg/ml were run in duplicatewithin the same run. The results were plotted to show assay linearity,and are shown in FIG. 4 . The assay is linear up to 20 μg/mL.

Example 7: Specimen Type Studies

Correlation studies were performed on spiked samples in differentspecimen types (serum, EDTA plasma, and Heparin plasma). The results ofthese studies are shown in Table 4.

TABLE 4 Comparison of Serum, EDTA Plasma and Heparin Plasma Sample TypesExpected Serum EDTA Plasma Heparin Plasma Value (μg/ % (μg/ % (μg/ %(μg/mL) mL) Recovery mL) Recovery mL) Recovery 2.63 2.64 100.32 2.73103.74 2.81 106.78 3.03 3.05 100.65 2.97 98.01 3.10 102.30 3.92 4.01102.26 3.93 100.22 3.95 100.73 4.44 4.60 103.50 4.42 99.45 4.97 111.835.56 5.77 103.86 6.12 110.16 5.81 104.58 5.97 6.09 102.01 6.28 105.196.12 102.51 6.45 6.78 105.09 6.67 103.39 6.82 105.71 7.02 7.20 102.607.48 106.59 7.34 104.60 8.51 8.95 105.16 9.09 106.81 8.76 102.93 9.099.60 105.60 10.40 114.40 9.94 109.34 10.81 11.00 101.75 11.10 102.6811.50 106.38 12.50 13.10 104.80 13.40 107.20 13.60 108.80 13.33 13.60102.00 13.50 101.25 13.50 101.25 14.81 15.00 101.25 15.50 104.63 15.00101.25 18.18 18.70 102.85 18.50 101.75 19.20 105.60

Results are presented graphically in FIG. 5 . Serum data exhibited a R²value of 0.9991; EDTA Plasma exhibited a R² value of 0.9949; and HeparinPlasma exhibited a R² value of 0.9965. Thus, all tested specimen typesare acceptable for analysis.

Example 8: Recovery

A recovery study of lacosamide in spiked serum was performed (inquadruplicate for the seven concentrations described in Example 6).Recovery of the QC material was acceptable for all concentrations tested(bias<TEa/4, assay slope=0.9710, and r=0.9994).

Example 9: Interference

An interference study was performed by introducing differentconcentrations of hemoglobin, triglycerides, and bilirubin into serumsamples spiked with 2 μg/mL and 1.6 μg/mL of lacosamide. The sampleswere then subjected to HPLC-MS/MS as described in Example 3. Hemoglobin,triglycerides, and bilirubin did not significantly interfere withmeasurement of lacosamide in any of the samples tested. Tables 5 and 6list the results for the interference study.

TABLE 5 Interference of Hemoglobin, Triglycerides, and Bilirubin withDetecting 2.0 μg/mL Lacosamide in Serum 1st Result 2nd Result AverageRecovery (μg/mL) (μg/mL) (μg/mL) % Hemoglobin Neat 1.91 1.85 1.88 High2.04 2.02 2.03 107.98 Medium 2.03 1.96 2.00 106.12 Low 1.99 2.00 2.00106.12 Triglycerides Neat 1.91 1.85 1.88 High 1.83 1.85 1.84 97.87Medium 1.90 1.97 1.94 102.93 Low 1.99 2.04 2.02 107.18 Bilirubin Neat1.91 1.85 1.88 High 1.92 1.92 1.92 102.13 Medium 2.10 2.07 2.09 110.90Low 2.03 1.99 2.01 106.91

TABLE 6 Interference of Hemoglobin, Triglycerides, and Bilirubin withDetecting 1.6 μg/mL Lacosamide in Serum 1st Result 2nd Result AverageRecovery (μg/mL) (μg/mL) (μg/mL) % Hemoglobin Neat 1.48 1.49 1.49 High1.60 1.59 1.60 107.41 Medium 1.60 1.64 1.62 109.09 Low 1.58 1.67 1.63109.43 Triglycerides Neat 1.48 1.49 1.49 High 1.45 1.36 1.41 94.61Medium 1.57 1.52 1.55 104.04 Low 1.60 1.60 1.60 107.74 Bilirubin Neat1.48 1.49 1.49 High 1.55 1.61 1.58 106.40 Medium 1.58 1.57 1.58 106.06Low 1.57 1.59 1.58 106.40

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining the amount oflacosamide in a sample, said method comprising: (i) purifying lacosamidefrom said sample by high performance liquid chromatography (HPLC); (ii)ionizing lacosamide to produce at least two lacosamide fragment ionscomprising a quantifier ion and a qualifier ion detectable by massspectrometry; (iii) determining the amount of lacosamide by measuringthe amount of a quantifier ion and confirming the identity of thelacosamide ion by detecting a qualifier ion by mass spectrometry.
 2. Themethod of claim 1, wherein said quantifier ion has a mass/charge ratioof 91.0±0.5.
 3. The method of claim 1, wherein said qualifier ion has amass/charge ratio of 108.0±0.5.
 4. The method of claim 1, wherein saidmass spectrometry is tandem mass spectrometry.
 5. The method of claim 1,wherein said sample is plasma.
 6. The method of claim 1, wherein saidsample is serum.
 7. The method of claim 1, wherein said ionizing is byelectrospray ionization.
 8. The method of claim 1, wherein said ionizingis conducted in positive ion mode.